Normal brain function requires proper neuronal connections. Layer 2/3 cortical pyramidal neurons in a mammalian brain have similar morphological and functional characteristics, but they tend to form functionally distinct microcircuits by making specialized connections. The cellular and molecular mechanisms by which one neuron finds specific target neurons and eventually forms functional subnetworks are not fully understood. A growing body of evidence indicates that the functional microcircuit formation is largely influenced by the activity pattern arising in local circuits. Presumably, the exact timing of action potential firing, the degree of excitability, and the level of inhibition ar important, but how these factors are operated together and are expressed at the level of multiple neurons have not yet been precisely defined. We propose to address these questions by using electrophysiological and optical approaches to visualize and manipulate neuronal activities at individual cell level. In the Aim 1, we seek to determine how spikes generated in multiple neurons within a short time window influence circuit reorganization by varying three factors: spike timing and number, distance between neurons, and the number of neurons. In Aim 2, cellular mechanisms such as neuronal excitability and the involvement of disinhibition will be examined. Lastly, Aim 3 is designed to test whether circuit assembly can be manipulated in awake behaving animals. Completion of the proposed work will provide mechanistic insight during cortical circuit plasticity and would establish experimental evidence for non-random features of neural connectivity in the mammalian brain. Abnormal neuronal connectivity has been implicated in various neuropsychiatric diseases such as schizophrenia, epilepsy, and autism spectrum diseases, and can directly influence the symptoms of other brain disorders or injuries. Thus, understanding cellular mechanisms of activity-dependent redistribution of local circuits could also help inform the development of novel strategies for circuit dysfunction.